Molecular Genetics

Historical Context of DNA as Genetic Material

  • Key Studies in Heredity:

    • 1865, Gregor Mendel: Studies in heredity

    • 1869-90s, Johann Friedrich Miescher: Analysis of nuclei contents

    • Early 20th Century, Thomas Hunt Morgan: Hypothesized that genes are located on chromosomes

    • Essential Question: The nature of hereditary material: proteins or DNA?

Griffith’s Transformation (1927)

  • Study Focus: Bacteria causing pneumonia

  • Observations Made:

    • Virulent Strains:

    • Encapsulated, forms smooth colonies on agar

    • Avirulent Strains:

    • Lack a capsule, forms rough colonies on agar

  • Key Experiment:

    • Heat-killed virulent strains can transform avirulent strains into virulent strains

The Transforming Principle is DNA

  • Key Researchers: Avery, MacLeod, and McCarty (1944)

  • Experiment:

    • Utilized DNase (deoxyribonuclease) to destroy the transforming activity.

    • Conclusion: DNA is the transforming principle, not proteins.

Hershey and Chase (1952)

  • Organisms Used: Escherichia coli and bacteriophage T2

  • Techniques: Used radioisotopes 32P (phosphorus) and 35S (sulfur)

  • Findings:

    • Demonstrated that DNA enters bacterial cells during infection and directs viral reproduction.

Four Macromolecules

  • Types:

    • Carbohydrates: Composed of carbon (C), hydrogen (H), oxygen (O)

    • Lipids: Composed of carbon (C), hydrogen (H), oxygen (O)

    • Nucleic Acids: Composed of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus (P)

    • Proteins: Composed of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), sulfur (S)

The Building Blocks of DNA

  • Components:

    • Nitrogenous Base

    • Pentose Sugar: Deoxyribose

    • Phosphate Group

    • Note: A nucleoside lacks a phosphate group; RNA is ribonucleic acid whereas DNA is deoxyribonucleic acid.

Nitrogenous Bases

  • DNA Bases:

    • Adenine (A)

    • Cytosine (C)

    • Guanine (G)

    • Thymine (T)

  • RNA Bases:

    • Adenine (A)

    • Cytosine (C)

    • Guanine (G)

    • Uracil (U)

DNA Strands

  • Bonding:

    • Two mononucleotides link via a phosphodiester bond between the C3’ and C5’ positions

    • A DNA structure has a free 5’ end and a free 3’ end.

Structure of DNA (Erwin Chargaff, 1950s)

  • Findings:

    1. The amount of adenine (A) is proportional to the amount of thymine (T); the amount of guanine (G) is proportional to the amount of cytosine (C)

    2. The sum of purines (A, G) is roughly equal to the sum of pyrimidines (C, T)

    3. The percentage of guanine (G) and cytosine (C) varies and is not equivalent to the percentage of adenine (A) and thymine (T)

  • Example: If 15% of the nitrogenous bases in a DNA sample are thymine, what percentage of cytosine would be expected?

    • Answer: 15% thymine implies 15% adenine, leaving 70% for G and C, therefore: (G + C = 70 ext{%}), G
      eq C

The Structure of DNA: Watson and Crick Model (1953)

  • Characteristics:

    • Right-handed double helix made of two anti-parallel strands

    • Major and minor grooves are present

    • Phosphate-sugar backbone on the outside, bases stacked within

    • One turn of the helix measures approximately 3.4 nm

    • Diameter of the double helix is about 2 nm

DNA Base Pairing

  • Pairing Rules:

    • Purine + Pyrimidine: Stable structure

    • Examples: A-T and G-C pairings

    • Incorrect pairings (e.g., Pyrimidine + Pyrimidine or Purine + Purine) result in either too skinny or too wide DNA structure

Major and Minor Grooves

  • Definition: Two distinct sizes of grooves running in a spiral

    • Major grooves: Shallow

    • Minor grooves: Deep

DNA Structure: Genetic Material Requirements

  1. The double-helical structure indicates a genetic code, written as sequences of nucleotides and translated into amino acids during protein synthesis.

  2. The specific base pairing suggests a possible mechanism for copying genetic material.

  3. Mutations can occur through the substitution of nucleotides at different positions.

Proposed Models of DNA Replication

  • Models:

    1. Conservative Model

    2. Semiconservative Model

    3. Dispersive Model

Semiconservative DNA Replication

  • Experiments:

    • Meselson and Stahl (1958): Used 15N-labeled E. coli grown in 14N medium

    • Result: Each new DNA molecule consists of one old strand (15N) and one newly synthesized strand (14N), providing strong evidence for semiconservative replication in prokaryotes.

Visuals from Semiconservative Replication Experiment

  • Outcomes:

    • First generation showed a hybrid of heavy and light DNA

    • Subsequent generations further clarified the patterns observed based on model predictions

Visual Representation of DNA Structure

the theoretical implications showing both heavy (15N) and light (14N) labeled DNA strands across generations

Repeating Patterns of Semiconservative Replication

  • Example Sequences:

    • Original strands:

    • 5'-ACTATCCGCATG-3'

    • 3'-TGATAGGCGTAC-5'

    • Result: Produces complementary strands, with corresponding ends maintained in the replication process.